High-resolution video microscopy techniques demonstrate that filopodia and lamellipodia which develop during platelet activation are highly dynamic structures. We seek to understand how the cytoskeletal processes that give rise to these morphological dynamics are regulated. The four specific aims of this project are: 1. Comprehensive characterization of the morphological dynamics of platelet activation. The effects of weak and strong agonists, shear forces, well characterized platelet inhibitors and intrinsic platelet defects will be examined, using video-enhanced differential interference contrast microscopy, in order to establish baseline characteristics for study of platelets by this novel technique. 2. How does the substrate for platelet activation affect the cytoskeletal transformation and motility of platelets? Platelet activation on physiologically relevant substrates such as endothelial cells and purified subendothelial extracellular matrix proteins will be studied. 3. Do localized subcellular changes in platelet calcium concentration regulate the platelet cytoskeletal transformation? Single-cell calcium imaging, in conjunction with simultaneous differential interference contrast imaging of platelets, will be used to correlate spatial and temporal changes in intracellular calcium with the morphological changes associated with platelet activation. 4. Is gelsolin required for regulation of the platelet cytoskeleton? Platelets from gelsolin "knockout" mice will be analyzed by video microscopy techniques, to determine the extent to which this actin filament-severing protein contributes to the morphological transformation of platelets. The results of these experiments should provide answers to important questions about platelet physiology which are of particular relevance to hemostasis and vascular medicine. The proposal bridges the principal investigator's background in cytoskeletal biochemistry with his training in Hematology and will serve to further his development of an independent research career.